Nicotinic receptor agonists for the treatment of inflammatory diseases

ABSTRACT

Nicotine receptor agonists or analogs or derivatives thereof for treating inflammatory pulmonary diseases, and pharmaceutical compositions including nicotine receptor agonists or analogs or derivatives thereof. Compounds of formula wherein R1, R2, Xa and Ya are as defined herein are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/632,051, filed on Jan. 10, 2007, which is a national stage entry ofApplication No. PCT/CA2005/001120, filed on Jul. 15, 2005, which is acontinuation-in-part of U.S. application Ser. No. 10/890,987 filed Jul.15, 2004. The entire contents of each of U.S. application Ser. No.11/632,051, Application No. PCT/SE00/00785 and U.S. application Ser. No.10/890,987 are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to the treatment of inflammatory diseases,including a variety of pulmonary diseases, through the use oradministration of nicotinic receptor agonists or analogs and derivativesthereof.

b) Description of Prior Art

Although a normal man or woman breathes more than one cubic meter of airevery hour, our lung defense mechanisms usually deal with the largequantities of particles, antigens, infectious agents and toxic gases andfumes that are present in inhaled air. The interaction of theseparticles with the immune system and other lung defense mechanismsresults in the generation of a controlled inflammatory response which isusually protective and beneficial. In general, this process regulatesitself in order to preserve the integrity of the airway and alveolarepithelial surfaces where gas exchange occurs. In some cases, however,the inflammatory response cannot be regulated and the potential fortissue injury is increased. Depending on the type of environmentalexposure, genetic predisposition, and a variety of ill-defined factors,abnormally large numbers of inflammatory cells can be recruited atdifferent sites of the respiratory system, resulting in illness ordisease.

The inflammatory response to inhaled or intrinsic stimuli ischaracterized by a non-specific increase in the vascular permeability,the release of inflammatory and chemotactic mediators includinghistamine, eicosanoids, prostaglandins, cytokines and chemokines. Thesemediators modulate the expression and engagement ofleukocyte-endothelium cell adhesion molecules allowing the recruitmentof inflammatory cells present in blood.

A more specific inflammatory reaction involves the recognition and themounting of an exacerbated, specific immune response to inhaledantigens. This reaction is involved in the development of asthma,hypersensitivity pneumonitis (HP) and possibly sarcoidosis.Dysregulation in the repair mechanisms following lung injury maycontribute to fibrosis and loss of function in asthma, pulmonaryfibrosis, chronic obstructive pulmonary disease (COPD), and chronic HP.

It was previously reported that the incidence of HP is much lower amongcurrent smokers than in non-smokers (1-4). Sarcoidosis is also lessfrequent in smokers than in non smokers (5, 6), The mechanismsunderlying the beneficial effects of cigarette smoking on thedevelopment of HP and other inflammatory diseases are still unknown butmay be linked to the immunomodulatory effect of nicotine. There areclinical observations of asthma de novo or exacerbation after smokingcessation. Proof of this is difficult to obtain and any protectiveeffects of nicotine in the prevention or treatment of asthma are likelyoverwhelmed by the negative effects of tobacco smoke with its thousandsof constituents.

The protective effect of smoking has also been reported in otherdiseases, the most studied being ulcerative colitis, an inflammatoryintestinal disease (7, 8). Nicotine has been successfully used in thetreatment of this disease (9, 10). Other studies have looked at thepossible therapeutic value of nicotine in the treatment of Alzheimer'sdisease and Parkinson's disease. (11, 12).

Nicotinic receptors are pentamers made up of five polypeptide subunitswhich act as ligand-gated ions channels. When the ligand binds to thereceptor, a conformational change in the polypeptide occurs, opening acentral channel that allows sodium ion to move from the extracellularfluid into the cytoplasm. Four types of subunits have been identified:α, β, γ and δ. The receptor can consist of any combination of these fourtypes of subunits (13). Recent work has shown that alveolar macrophages(AM) can express the α-7 subunit (14), while bronchial epithelial cellsexpress the α-3, α-5 and α-7 subunits (15), and lymphocytes the α-2,α-5, α-7, β-2 and β-4 subunits (14). Fibroblasts (16) and airway smoothmuscles cells (17) also express these receptors. Therefore, residentpulmonary cells (AM, dendritic cells, epithelial cells, fibroblasts,etc.) and those recruited in inflammatory diseases (lymphocytes,polymorphonuclear cells) express nicotinic receptors.

Nicotinic receptor activation in lymphocytes affects the intracellularsignalization, leading to incomplete activation of the cell. In fact,nicotine treatment upregulates protein kinase activity, which in turnupregulates phospholipase A2 (PLA2) activity. PLA2 is responsible forcleaving phosphoinositol-2-phosphate (PIP2) into inositol-3-phosphate(IP3) and diacylglycerol (DAG) (18, 19). The continuous presence of IP3in the cell would appear to result in the desensitization of calciumstores, leading to their depletion (19). This observation could explainthe fact that nicotine-treated lymphocytes do not release enough calciuminto the cytoplasm to activate transcription factors such as NFk-B (20).

Nicotine, the major pharmacological component of cigarette smoke, is oneof the best known nicotinic receptor agonists (21). This naturalsubstance has well defined anti-inflammatory and immunosuppressiveproperties (22), and may have anti-fibrotic properties (23). Exposure ofanimals to smoke from cigarettes with high levels of nicotine is moreimmunosuppressive than that from low-nicotine cigarettes (24). Moreover,treatment of rats with nicotine inhibits the specific antibody responseto antigens and induces T cell energy (25). Although they are increasedin number, AM from smokers show a decreased ability to secreteinflammatory cytokines in response to endotoxins ((20, 25, 26)) andnicotine seems, to be the responsible component of this inhibition (26).One study also showed that peripheral blood lymphocytes from smokersexpress higher levels of FAS ligand (FASL) and that nicotine increasesFASL expression on lymphocytes from non-smokers, indicating thatnicotine may affect cell apoptosis (27). Nicotine was also shown to havean inhibitory effect on the proliferation and extracellular matrixproduction of human gingival fibroblasts in vitro (23). Of interest,nicotine treatment seems to up-regulate the expression of nicotinicreceptors (28). Nicotine itself is a safe substance that does not seemto have any long term side effects (48-49). Smoke-related diseases ofthe lungs, heart and arteries are not caused by nicotine but by thethousands of other chemicals present in the inhaled smoke. The mainproblem is that nicotine crosses the blood-brain barrier, inducingaddiction. The harmful effects of cigarette smoking are obvious.Although nicotine is not responsible for the toxic effects of cigarettesmoking, the association remains.

Nicotinic agonists may down-regulate T cell activation, indeed, nicotinehas been shown to affect T cell expression of the co-stimulatorymolecules CD28 and CTLA4 (29).

The B7/CD28/CTLA4 co-stimulatory pathway plays a key regulatory role inT-cell activation and homeostasis (30, 31). Two signaling pathways areinvolved. A positive signal involves the engagement of B7 (CD80/CD86)molecules, with T cell CD28 receptors which results in the potentiationof T cell responses (proliferation, activation, cytokine expression, andsurvival) (32). A negative signal involves B7 interactions with CTLA4 onactivated T cells, leading to a downmodulation of T cell responses (33,34). The balance between CD28 and CTLA4 derived signals may alter theoutcome of T-cell activation.

In HP, it was previously reported that an upregulation of B7 moleculeexpression on AM in patients with active HP (35) and in murine HP (36).It was also shown that a blockade of the B7-CD28 co-stimulatory pathwayin mice inhibited lung inflammation (36). These results alsodemonstrated that the expression of B7 molecules on AM is lower insmokers than in non-smokers and that an in vitro influenza virusinfection is able to upregulate B7 expression in normal human AM but notin AM from smokers; whether this is due to nicotine or other substancespresent in cigarette smoke is unknown (35). An up-regulation of the B7molecules has also been reported in asthma (37, 38) and sarcoidosis(39).

Epibatidine is the most potent nicotinic agonist known so far (40). Ithas anti-inflammatory and analgesic properties. In fact, its analgesicpotential is two hundred times that of morphine (40). This molecule isalso known to inhibit lymphocyte proliferation in vitro (41). Thebinding of epibatidine to the receptor is non-specific (42).Unfortunately, epibatidine has major toxic side effects mostly on thecardiovascular and the central nervous systems making it inappropriatefor use as an anti-inflammatory drug to treat pulmonary diseases (40).

Dimethylphenylpiperazinium (DMPP) is a synthetic nicotinic agonist thatis non-specific (13). Its potency for the receptor is about the same asnicotine, depending on the kind of cells implicated in the stimulation(43). Its advantage over nicotine and other nicotinic agonists is thatits chemical configuration prevents it from crossing the blood-brainbarrier, thus causing no addiction or other central nervous effects(13). The anti-inflammatory properties of DMPP are not well described.However, it has been shown that a chronic in vivo treatment coulddecrease the number of white, blood cells, decrease the cytokineproduction by splenocytes and decrease the activity of natural killercells (44). The effect of DMPP on airway smooth muscle cells has alsobeen tested. DMPP has an initial short contractive effect which isfollowed by a relaxing effect when the cells are in contact with theagonist for a longer period of time (45). This bronchodilatory effectmay not necessarily in itself make DMPP the most useful treatment ofasthma, since other potent bronchodilators are currently available onthe market (B2 agonists). However, the properties of this nicotinicreceptor agonist are important since this drug could be safelyadministered to asthmatics and COPD patients for its anti-inflammatoryproperties. Moreover, there is no apparent evidence that DMPP has anytoxic effect on major organs such as the heart, the brain, the liver orthe lungs.

Corticosteroids are potent anti-inflammatory drugs. Their systemic usecauses major side effects that preclude their long-term uses wheneverpossible. Inhaled poorly absorbed steroids are useful to treat airwayinflammation. At low doses these drugs have little or no side effects.However, higher doses increase the risks for oral candidasis, vocalcords paralysis, cataracts and osteoporosis. Inhaled steroids have noeffects on lung interstitium and have no anti-fibrotic properties (57)

More recent drugs, such as anti-leukotrienes, are useful in someasthmatics (58) but have no effects in COPD and other lung diseases.These drugs have anti-inflammatory properties limited to the componentsof inflammation caused by leukotrienes (59). The treatment ofinterstitial lung disease such as IPF, Sarcoidosis, HP, and BOOPbasically rests on the use of systemic corticosteroids. This treatmentis effective in controlling some of the inflammation but unfortunatelyinduces serious side effects and does not reverse underlying fibroticchanges. Immunosupressive agents such as cyclophosphamide andazathioprine are sometimes tried in severe IPF but their therapeuticvalues are unproven and at most, very limited (60). In essence, lungfibrosis is usually progressive and untreatable, with most IPF patientsdying of this condition (61).

Despite advances in the treatment of inflammatory illnesses, includingpulmonary inflammatory diseases, treatment using available drugs oragents frequently results in undesirable side effects. For example, theinflammation of COPD is apparently resistant to corticosteroids, andconsequently the need for the development of new anti-inflammatory drugsto treat this condition has been recognized (46).

Similarly, while corticosteroids and other immunosuppressive medicationshave been routinely employed to treat pulmonary fibrosis, they havedemonstrated only marginal efficacy (47).

There is thus a need for new and reliable methods of treatinginflammatory diseases, including pulmonary inflammatory diseases, in amanner that alleviates their symptoms without causing side effects.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novelmethod for treating inflammatory diseases. Specifically, a novel methodis described for treating pulmonary inflammatory diseases through theuse or administration of an agent that binds to or modulates thefunction nicotinic receptor, such as nicotinic receptor agonists oranalogues or derivatives thereof.

In one aspect, there is therefore provided a method for treating orpreventing pulmonary inflammatory diseases comprising administering aneffective amount of a compound that modulates the function of nicotinicreceptors.

In a further aspect, there is also provided compounds of formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N,Ya is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, aralkyl, aryl of 6 to 10 carbon atomsand 3 to 10 membered heterocyclen is an integer from 0 to 2,J is a counter ion.

In still a further aspect, there is provided a pharmaceuticalcomposition for treating pulmonary inflammatory diseases comprising anicotinic receptor agonist and a pharmaceutically acceptable excipient.

In a further aspect, there is provided a method for inducing airwayssmooth muscle relaxation comprising administering an effective amount ofa compound having the formula:

wherein R₁, R₂, Xa, Ya and J are as described herein.

In another aspect, there is provided by the present invention a methodfor inducing agonistic response in a pulmonary cell nicotinic receptor,comprising administering an effective amount of a nicotinic receptoragonist.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated but is not limited by the annexed drawings,in which:

FIG. 1 shows total and differential cell counts in BAL cells;

FIG. 2 shows IFN-γ mRNA expression in isolated lung mononuclear cells;

FIG. 3 illustrates TNF-α mRNA expression induced by a 24 h LPSstimulation;

FIG. 4 illustrates TNF-α mRNA expression induced by a 24 h SRstimulation;

FIG. 5 illustrates IL-10 mRNA expression induced by a 24 h LPSstimulation;

FIG. 6 illustrates IL-10 mRNA expression induced by a 24 h SRstimulation. nicotine treatment occurred at 160 μM (60% drop ofexpression), and at 80 μM (90% drop of expression) with the DMPPtreatment;

FIG. 7 illustrates IFN-γ mRNA expression induced in RAW 264.7 cells by a24 h LPS stimulation;

FIGS. 8 (a) and (b) show the expression of CD 80 induced with either LPS(38%) or SR antigen (35%);

FIG. 9 illustrates IFN-γ mRNA expression in T lymphocytes isolated fromBAL performed on HP patients;

FIG. 10 illustrates CD 86 expression in total cells from a BAL that wasperformed on a normal patient;

FIG. 11 illustrates BAL cells from DMPP, nicotine and epibatidinetreated mice;

FIG. 12 illustrates a significant inhibitory effect of DMPP on lunginflammation was found when increasing the number of animals;

FIG. 13 illustrates TNF levels in BAL fluid from DMPP-treated mice;

FIG. 14 illustrates the effect of intra-peritoneal treatment withincreasing doses of DMPP on total cell accumulation in BAL of asthmaticmice;

FIG. 15 illustrates differential counts for the dose response;

FIG. 16 illustrates the second dose response for the DMPP IP treatmenteffect on total cell accumulation in BAL of asthmatic mice;

FIG. 17 illustrates differential counts from the second dose response;

FIG. 18 illustrates BAL IL-5 levels from control, asthmatic and treatedmice;

FIG. 19 illustrates lung resistance after metacholine challenges fromnormal, asthmatic and asthmatic treated with 0.5 mg/kg intranasal DMPP;

FIG. 20 illustrates a calculation of the provocative challenge dose of200% lung resistance augmentation (PC 200);

FIG. 21 illustrates IL-4 mRNA expression induced by a 24 h LPSstimulation;

FIG. 22 illustrates the effect of DMPP on blood eosinophiltransmigration;

FIG. 23 illustrates the effect of mecamylamine, a nicotinic antagonist,on the inhibitory effect of DMPP on blood eosinophil transmigration;

FIG. 24 illustrates the effect of additional nicotinic agonists(nicotine, epibatidine and cytisine) on transmigration of bloodeosinophils;

FIG. 25 illustrates the effect of DMPP on collagen 1A mRNA expression bynormal human lung fibroblasts;

FIG. 26 illustrates the effect of nicotine on collagen 1A mRNAexpression by human lung fibroblasts;

FIG. 27 illustrates the effect of epibatidine, another nicotinicagonist, on collagen 1A mRNA expression by human lung fibroblasts;

FIG. 28 illustrates the effect of DMPP, ASM-002, ASM-003, ASM-004, andASM-005 on TNF release

FIG. 29 illustrates the effect of DMPP, ASM-002, ASM-003, ASM-004, andASM-005 on mouse tracheal airway smooth muscle responsiveness;

FIG. 30 illustrates the effect of ASM-002 on lung inflammation;

FIG. 31 illustrates the effects of ASM-002 on lung resistance in a mousemodel of asthma;

FIG. 32 illustrates the comparative effects of ASM-002 and prednisone onlung inflammation;

FIG. 33 illustrates the effects of ASM-002 in a dog model of lunghyper-responsiveness;

FIG. 34 illustrates the muscle-relaxing properties of ASM-002 on mousetracheas;

FIG. 35 illustrates the muscle-relaxing properties of ASM-002 on dogbronchial rings;

FIG. 36 illustrates the muscle-relaxing properties of ASM-002 on humanbronchial rings;

FIG. 37 illustrates the inhibitory effects of ASM-002 on potentinflammatory mediators release by human blood cells isolated fromasthmatic patients;

FIG. 38 illustrates the comparative effects of ASM-002 with DMPP anddexamethasone on TNF production by LPS-stimulated blood monocytes;

FIG. 39 illustrates the inhibition of LTC4 production by ASM-002;

FIG. 40 illustrates the effect of nicotine, ASM-N1, ASM-N2, ASM-N3,ASM-N4 and ASM-002 on TNF production;

DESCRIPTION OF PREFERRED EMBODIMENTS

Other objects, advantages and features of the present invention willbecome more apparent upon reading the following non-restrictivedescription of preferred embodiments thereof, given by way of example,only with reference to the accompanying drawings.

The idea of using nicotine or other nicotinic receptor agonists oranalogs or derivatives thereof to treat inflammatory pulmonary diseaseis novel. Despite the impressive anti-inflammatory and immunosuppressiveproperties of nicotine and other nicotinic receptor agonists or analogsor derivatives, their usefulness in the treatment of allergic and otherinflammatory lung diseases has not previously been disclosed. Thedrawbacks associated with cigarette are major reasons for the lack ofprior interest in nicotinic agonists or analogs or derivatives thereofin the treatment of lung diseases.

The present invention thus proposes the of use nicotinic receptoragonists, such as DMPP and analogs as well as derivatives thereof, totreat inflammatory lung diseases such as asthma, COPD, interstitialpulmonary fibrosis (IPF), sarcoidosis, HP, and bronchiolitis obliteranswith organizing pneumonitis (BOOP). The drug could be administeredorally or, depending on the specific diseases or conditions, by targeteddelivery directly to the lung by aerosolisation with different andpreferred vehicles in order to minimize systemic effects.

The anti-inflammatory, immunosuppressive and/or bronchodilatingproperties, as well as minimal side effects of nicotinic receptoragonists and analogs and derivatives thereof, make these drugs ideallysuited for medical use in the treatment of a large variety of lungdiseases that are characterized by bronchial or Interstitialinflammation. These diseases include diseases such as asthma, COPD, IPF,sarcoidosis, HP and BOOP.

In accordance with one embodiment, the invention provides a method fortreating or preventing pulmonary inflammatory diseases comprisingadministering an effective amount of a compound that modulates thefunction of nicotinic receptors.

In one embodiment, the method is useful for treating pulmonaryinflammatory diseases.

In one embodiment, the compound for use in the method of the inventionis a nicotinic receptor agonist.

In one embodiment, the nicotinic receptor agonists is selected from thegroup consisting of dimethylphenylpiperazinium (DMPP), nicotine,epibatidine, cytisine, acetylcholine, and analogs thereof.

In another embodiment, the compounds for use in the method of theinvention are:

i) a compound having the formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N,Ya is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, aralkyl, aryl of 6 to 10 carbon atomsand 3 to 10 membered heterocyclen is an integer from 0 to 2,J is a counter ion;or ii) a compound having the formula:

wherein R₃ is selected from

Xb is N or N⁺—R₁₀,R₄ is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms,alkylthio of 1 to 10 carbon atoms, alkylamino of 1 to 10 carbon atoms,alkanol of 1 to 10 carbon atoms, aralkyl, aryl of 6 to 10 carbon atoms;each of R₁₀, R₁₁ and R₁₂ are independently, alkyl of 1 to 10 carbonatoms,provided that a counterion is present when Xb is N⁺—R₁₀;or iii) a compound having the formula:

wherein Xc is NR₁₃ or N⁺—R₁₃R₁₄, wherein R₁₃ and R₁₄ are independentlyalkyl of 1 to 10 carbon atoms,R₅ is a 3 to 10 membered heterocycle,provided that a counterion is present when Xc is N⁺—R₁₃R₁₄;or iv) a compound having the formula:

wherein W is O or S;each of Yc and Yd are independently selected from hydrogen, halogen,amino, amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso,urea, sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate,acyl, acyloxy, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbonatoms, alkylthio of 1 to 10 carbon atoms, alkylamino of 1 to 10 carbonatoms, alkanol of 1 to 10 carbon atoms, aralkyl, aryl of 6 to 10 carbonatoms;wherein Xd is NR₁₅ or N⁺—R₁₅R₁₆, wherein R₁₅ and R₁₆ are independentlyalkyl of 1 to 10 carbon atoms,provided that a counterion is present when Xd is N⁺—R₁₅R₁₆.

In a further embodiment, the compound useful in the method of theinvention has the formula:

wherein R₁ and R₂ are independently alkyl of 1 to 10 carbon atoms,Xa is CH or N,Ya is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms,alkylthio of 1 to 10 carbon atoms, alkylamino of 1 to 10 carbon atoms,alkanol of 1 to 10 carbon atoms, aralkyl, aryl of 6 to 10 carbon atomsand 3 to 10 membered heterocycle;n is an integer from 0 to 2,J is a counter ion.

In still a further embodiment, R₁ and R₂ are independently optionallysubstituted lower alkyl of 1 to 10 carbon atoms;

Xa is CH;

Ya is one or more substituent selected from hydrogen, halogen, amino,amido, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbonatoms and alkanol of 1 to 6 carbon atoms;

n is 1 or 2;

J is a halogen.

In another embodiment, the compounds for use in the method of theinvention has the formula:

wherein R₁ and R₂ are independently optionally substituted lower alkylof 1 to 6 carbon atoms;Xa is CH;Ya is one or more substituent selected from hydrogen, halogen, amino,amido, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbonatoms, lower alkanol of 1 to 6 carbon atoms;n is 1 or 2;J is a halogen.

In an additional embodiment, R₁ and R₂ are independently selected frommethyl, ethyl, n-propyl, or i-propyl;

Xa is CH;

Ya is hydrogen;

n is 1 or 2;

J is a halogen.

In an additional embodiment, the compound has the formula:

wherein R₁ and R₂ are independently selected from methyl, ethyl,n-propyl, or i-propyl;Ya is hydrogen;J is a halogen.

In a further embodiment, the compound for use in the method of theinvention has the formula:

In a further embodiment, the compound for use in the method of theinvention has the formula selected from:

In still a further embodiment, the compound for use in the method of theinvention has the formula selected from:

In one embodiment, the method according to the invention makes use of acompound that has the formula:

wherein R₃ is selected from

Xb is N or N⁺—N₁₀,R₄ is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms,alkylthio of 1 to 10 carbon atoms, alkylamino of 1 to 10 carbon atoms,alkanol of 1 to 10 carbon atoms, aralkyl, aryl of 6 to 10 carbon atoms;each of R₁₁ and R₁₂ are independently alkyl of 1 to 10 carbon atoms,provided that a counterion is present When Xb is N⁺—R₁₀.

In one embodiment, R₄ is one or more substituent selected from hydrogen,halogen, amino, amido, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of1 to 6 carbon atoms and alkanol of 1 to 6 carbon atoms; and R₁₁ and R₁₂are independently alkyl of 1 to 6 carbon atoms.

In a further embodiment, R₄ is one or more substituent selected fromhydrogen, and halogen; and R₁₁ and R₁₂ are independently alkyl of 1 to 6carbon atoms.

In a further embodiment, the compound for use in the method of theinvention has the formula selected from:

In one embodiment, the method according to the invention makes use of acompound that has the formula:

wherein Xc is NR₁₃ or N⁺—R₁₃R₁₄, wherein R₁₃ and R₁₄ are independentlyalkyl of 1 to 10 carbon atomsR₅ is a 3 to 10 membered heterocycle,provided that a counterion is present when Xc is N⁺—R₁₃R₁₄.

In one embodiment, R₁₃ and R₁₄ are independently alkyl of 1 to 6 carbonatoms.

In another embodiment, R₁₃ and R₁₄ are independently alkyl of 1 to 6carbon atoms; and R₅ is a 3 to 6 membered heterocycle.

In a further embodiment, R₁₃ and R₁₄ are independently alkyl of 1 to 6carbon atoms; and R₅ is an optionally substituted pyridyl.

In a further embodiment, the for use in the method of the invention hasthe formula selected from:

In one embodiment, the method according to the invention makes use of acompound that has the formula:

wherein W is O or S;each of Yc and Yd are independently a substituent selected fromhydrogen, halogen, amino, amidino, amido, azido, cyano, guanido,hydroxyl, nitro, nitroso, urea, sulfate, sulfite, sulfonate,sulphonamide, phosphate, phosphonate, acyl, acyloxy, alkyl of 1 to 10carbon atoms, alkoxy of 1 to 10 carbon atoms, alkylthio of 1 to 10carbon atoms, alkylamino of 1 to 10 carbon atoms, alkanol of 1 to 10carbon atoms, aralkyl, aryl of 6 to 10 carbon atoms;wherein Xd is NR₁₅ or N⁺—R₁₅R₁₆, wherein R₁₅ and R₁₆ are independentlyalkyl of 1 to 10 carbon atoms,provided that a counterion is present when Xd is N⁺—R₁₅R₁₆.

In one embodiment, Yc and Yd are independently one or more substituentselected from hydrogen, halogen, amino, amido, hydroxyl, alkyl of 1 to 6carbon atoms, alkoxy of 1 to 6 carbon atoms and alkanol of 1 to 6 carbonatoms.

In one embodiment, W is O; each of Yc and Yd are independently one ormore substituent selected from hydrogen, halogen, amino, amido,hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atomsand alkanol of 1 to 6 carbon atoms; and Xd is NR₁₅ or N⁺—R₁₅R₁₆, whereinR₁₅ and R₁₆ are independently alkyl of 1 to 6 carbon atoms.

In a further embodiment, W is O; each of Yc and Yd are independently oneor more substituent selected from hydrogen and halogen; and Xd is NR₁₅or N⁺—R₁₅R₁₆, wherein R₁₅ and R₁₆ are independently alkyl of 1 to 6carbon atoms.

In a further embodiment, the compound for use in the method of theinvention has the formula selected from:

In one embodiment, the pulmonary inflammatory disease is selected fromthe group consisting of asthma, chronic obstructive pulmonary disease(COPD), interstitial pulmonary fibrosis (IPF), sarcoidosis,hypersensitivity pneumonitis (HP), chronic HP and bronchiolitisobliterans with organizing pneumonitis (BOOP).

In one embodiment, the pulmonary inflammatory disease is selected fromthe group consisting of asthma, chronic obstructive pulmonary disease(COPD), interstitial pulmonary fibrosis (IPF), sarcoidosis,hypersensitivity pneumonitis (HP) and chronic HP.

In further embodiments, the pulmonary inflammatory disease is:

chronic obstructive pulmonary disease (COPD);

sarcoidosis;

hypersensitivity pneumonitis (HP).

In a further embodiment, the pulmonary inflammatory disease is asthma

In one embodiment of the invention, the compound for use in the methodof the invention is administered orally, parenterally, topically or byinhalation.

Alternatively, the compound is administered orally, topically, or byinhalation.

In one embodiment of the invention, the compound for use in the methodof the invention is administered orally.

In one embodiment, the compounds described herein are useful for themanufacture of a medicament for treating pulmonary inflammatorydiseases.

In one embodiment, there are novel compounds provided having theformula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N,Ya is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, aralkyl, aryl of 6 to 10 carbon atomsand 3 to 10 membered heterocyclen is an integer from 0 to 2,J is a counter ion.

In a further embodiment R₁ and R₂ are independently optionallysubstituted alkyl of 1 to 6 carbon atoms;

Xa is CH;

Ya is one or more substituent selected from hydrogen, halogen, amino,amido, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbonatoms and alkanol of 1 to 6 carbon atoms;

n is 1 or 2;

J is a halogen.

In one embodiment, the compound has the formula:

wherein R₁ and R₂ are independently optionally substituted alkyl of 1 to6 carbon atoms;X is CH;Y is one or more substituent selected from hydrogen, halogen, amino,amido, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbonatoms, alkanol of 1 to 6 carbon atoms;n is 1 or 2;J is a halogen.

In a further embodiment, R₁ and R₂ are independently selected frommethyl, ethyl, n-propyl, or i-propyl;

X is CH;

Y is hydrogen;

n is 1 or 2;

J is a halogen.

In an alternative embodiment, the compound has the formula:

wherein R₁ and R₂ are independently selected from methyl, ethyl,n-propyl, or i-propyl;Y is hydrogen;J is a halogen.

In still a further embodiment, the compound has the formula:

The first nicotinic receptor agonists include dimethylphenylpiperazinium(DMPP), nicotine, epibatidine, cytisine, acetylcholine and analoguesthereof.

Alternatively, nicotinic receptor agonists that can be used for thetreatments and uses according to the invention include the followingnicotinic receptor agonists and analogues thereof:

1-DMPP and analogs thereof

Compound R₁ R₂ X Y N DMPP CH₃ CH₃ CH — 1 CH₃ CH₂CH₂CH₃ CH — 1 or 2CH₂CH₃ CH₂CH₃ CH — 1 or 2 CH₂CH₃ CH₃ CH — 1 or 2 CH₃ CH₃ CH — 2 CH₃ — N— 1 H — N halogen 1

2- Nicotine and analogs

Position Compd X R₁ of R₁ R₂ Nicotine N

3 H N

3 H N

3 H N

4 H N

3 Halogen N

3 H N

3 H

3- Analogs of pyridylether

Position Compd X R₁ R₁ R₂ n O H —

1 O Aryl, alkyl, substituted- phenyl 5

1 O halogen 6

1 O H —

1, 2 or 3 R1 and R2 = alkyl, n = 1 or 2 NHC₃ H —

1, 2 or 3 R1 and R2 = alkyl, n = 1 or 2

4- Epibatidine and analogs

Compound R₁ R2 Epibatidi-ne

H X =halogen

H X =halogen

H

H

H or CH₃(alkyl) X =halogen

H or CH₃(alkyl) R1 and R2 =alkyl, n = 1 or 2

H or CH₃(alkyl) X = N⁺(CH₃)₃

5- Trimethaphan and analogs

Compond R X Trimethaphan

—

Halogen N⁺(CH₃)₃ — N⁺(CH2CH₃)₃ —

6- Cytisine and analogs

Compound R W X Y Z Cytisine H O H H H nBu O H H H H O halogen H halogenH S H H H (CH₃)₂ O or S halogen H halogen (CH₂CH₃)CH₃ O or S H H H(CH₂CH₃)₂ O or S H H H

7- Acetylcholine and analogs

Compound R Acetylcholine N⁺(CH₃)₃ N⁺(CH₂CH₃)₂CH₃ N⁺(CH₂CH₃)₃

8- N-methylcarbamylcholine and analogs

Compound R N- N⁺(CH₃)₃ methylcarbamylcoline * N⁺(CH₂CH₃)₂CH₃ *N⁺(CH₂CH₃)₃

9- ABT-418 and analogs

Compound R ABT-418 CH₃ (CH₃)₂ (CH₂CH₃)CH₃ (CH₂CH₃)₂

10- GTS-21 and analogs

Compound R₁ R₂ GTS-21 OCH₃ OCH₃ N⁺(CH₃)₃ OCH₃ OCH₃ N⁺(CH₃)₃

11- Arecoline and analogs

Compound R Arecoline CH₃ (CH₃)₂ (CH₂CH₃)CH₃ (CH₂CH₃)₂

12- Lobeline and analogs

Compound R Lobeline H (CH₃)₂ (CH₂CH₃)CH₃ (CH₂CH₃)₂

13- Analogs of philanthotoxin-433

Compound R n m NH₂ 4 3 N⁺(CH₃)₃ 1, 2, 3 or 4 1, 2 or 3 N⁺(CH₂CH₃)₂CH₃ 1,2, 3 or 4 1, 2 or 3 N⁺(CH₂CH₃)₃ 1, 2, 3 or 4 1, 2 or 3

14- Azabicyclic analogs

Compound R R n m

— 2 2

— 2 2

— 2 2

— 2 2

CH₃ 1 or 2 1 or 2

CH₃ 1 or 2 1 or 2

15- Analogs of SIB-1553

Compound R n CH₃ 1 (threo) CH₃ 0 (erythro) CH₃ 0 (threo) (CH₃)₂ 0 or 1(CH₂CH₃)CH₃ 0 or 1 (CH₂CH₃)₂ 0 or 1

16- Analogs of imidacloprit

Compound R X Y Z NO₂ Cl H NH H Cl N₃ S NO₂ Cl N₃ S N⁺(CH₃)₃ Cl H NH NO₂N⁺(CH₃)₃ H NH NO₂ Cl N⁺(CH₃)₃ NH

Of particular interest for the treatment of inflammatory pulmonarydiseases are the following analogues of DMPP, and having the formula:

in which R₁ is methyl or ethyl, R₂ is methyl, ethyl or propyl, X is CH,Y is hydrogen, n is 1 or 2.

The term “lower alkyl” represents a linear, branched or cyclichydrocarbon moiety having 1 to 10 carbon atoms and preferably 1 to 6carbon atoms, which may have one or more unsaturation in the chain, andis optionally substituted. Examples include but are not limited tomethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, isohexyl,neohexyl, allyl, vinyl, acetylenyl, ethylenyl, propenyl, isopropenyl,butenyl, isobutenyl, hexenyl, butadienyl, pentenyl, pentadienyl,hexenyl, hexadienyl, hexatrienyl, heptenyl, heptadienyl, heptatrienyl,octenyl, octadienyl, octatrienyl, octatetraenyl, propynyl, butynyl,pentynyl, hexynyl, cyclopropyl, cyclobutyl, cyclohexenyl,cyclohex-dienyl and cyclohexyl. The term “lower alkyl” is also meant toinclude alkyls in which one or more hydrogen atom is replaced by ahalogen, ie. an alkylhalide. Examples include but are not limited totrifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl,dichloromethyl, chloromethyl, trifluoroethyl, difluoroethyl,fluoroethyl, trichloroethyl, dichloroethyl, chloroethyl,chlorofluoromethyl, chlorodifluoromethyl, dichlorofluoroethyl.

The term “lower alkoxy” represents an alkyl which is covalently bondedto the adjacent atom through an oxygen atom. Examples include but arenot limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy,tert-pentyloxy, hexyloxy, isohexyloxy and neohexyloxy.

The term “lower Alkylthio” represents an alkyl which is covalentlybonded to the adjacent atom through a sulfur atom Examples include butare not limited to methylthio, ethylthio, propylthio, isopropylthio,butylthio, isobutylthio, sec-butylthio and tert-butylthio.

The term “lower Alkylamino” represents an alkyl which is covalentlybonded to the adjacent atom through a nitrogen atom and may bemonoalkylamino or dialkylamino, wherein the alkyl groups may be the sameor different. Examples include but are not limited to methylamino,dimethylamino, ethylamino, diethylamino, methylethylamino, propylamino,isopropylamino, butylamino, isobutylamino, sec-butylamino,tert-butylamino, pentylamino, isopentylamino, neopentylamino,tert-pentylamino, hexylamino, isohexylamino and neohexylamino,

The term “lower alkanol” represents an “alkyl” moiety for which one ofthe hydrogens has been replaced by an hydroxyl group. The term alkanolis also meant to include alkanol in which one or more hydrogen atoms isreplaced by a halogen. Examples include but are not limited to methanol,ethanol, propanol, isopropanol, butanol, ethyleneglycol,propyleneglycol, cyclopropanol or trifluoroethanol or fluoromethanol.

The term “aralkyl” represents an aryl group attached to the adjacentatom by a C₁₋₆ alkyl Examples include but are not limited to benzyl,benzhydryl, trityl, phenethyl, 3-phenylpropyl, 2-phenylpropyl,4-phenylbutyl and naphthylmethyl.

The term “aryl” represents a carbocyclic moiety containing at least onebenzenoid-type ring (i.e. may be monocyclic or polycyclic) having 6 to10 carbon atoms, and which may be optionally substituted with one ormore substituents. Alternatively, the ring may be containing 6 carbonatoms. Examples include but is not limited to phenyl, tolyl,dimethyphenyl, aminophenyl, anilinyl, naphthyl, anthryl, phenanthryl orbiphenyl,

The term “Acyl” is defined as a radical derived from a carboxylic acid,obtained by replacement of the —OH group. Like the acid to which it isrelated, an acyl radical may be straight chain, branched chain or cyclicaliphatic or aromatic. Examples include but are not limited to formyl,acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl,caproyl, isocaproyl, acryloyl, propioloyl, methacryloyl, crotonoyl,isocrotonoyl, benzoyl, naphthoyl, toluoyl, cinnamoyl, furoyl, glyceroyl,salicyloyl.

The term “Acyloxy” represents an acyl which is covalently bonded to theadjacent atom through an oxygen atom. Examples include but are notlimited to formyloxy, acetyloxy, propionyloxy, butyryloxy,isobutyryloxy, valeryloxy, isovaleryloxy, pivaloyloxy, caproyloxy,isocaproyloxy, acryloyloxy, propioloyloxy, methacryloyloxy,crotonoyloxy, isocrotonoyloxy, benzoyloxy, naphthoyloxy, toluoyloxy,hydroatropoyloxy, atropoyloxy, cinnamoyloxy, furoyloxy, glyceroyloxy,tropoyloxy, benziloyloxy, salicyloyloxy, anisoyloxy, vanilloyloxy,veratroyloxy, piperonyloyloxy, protocatechuoyloxy and galloyloxy, withpreference given to formyloxy; acetyloxy, propionyloxy, butyryloxy,isobutyryloxy, valeryloxy, isovaleryloxy, pivaloyloxy, benzoyloxy andnaphthoyloxy.

The term “halogen atom” is specifically a fluoride atom, chloride atom,bromide atom or iodide atom.

The term “counterion” is meant to include ion that accompanies an ionicspecies in order to maintain electric neutrality. Examples of counterionas used herein include but are not limited to fluoride, chloride,bromide, iodide, sulfate, sulfonate.

The term “independently” means that a substituent can be the same or adifferent definition for each item.

The term “heterocycle” represents a 3 to 10 membered optionallysubstituted, saturated, unsaturated or aromatic cyclic moiety whereinsaid cyclic moeity is interrupted by at least one heteroatom selectedfrom oxygen (O), sulfur (S) or nitrogen (N). Alternatively, heterocyclesmay be 3 to 6 membered ring or 5 to 6 membered ring. Heterocycles may bemonocyclic or polycyclic rings. Examples include but are not limited toazepinyl, aziridinyl, azetyl, azetidinyl, diazepinyl, dithiadiazinyl,dioxazepinyl, dioxolanyl, dithiazolyl, furanyl, isooxazolyl,isothiazolyl, morpholinyl, morpholino, oxetanyl, oxadiazolyl, oxiranyl,oxazinyl oxazolyl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl,piperidyl, piperidino, pyridyl, pyranyl, pyrazolyl, pyrrolyl,pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl,thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl, thiazinyl andthiopyranyl, furoisoxazolyl, imidazothiazolyl, thienolsothiazolyl,thienothiazolyl, imidazopyrazolyl, cyclopentapyrazolyl, pyrrolopyrrolyl,thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl,thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl,oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl,imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl,benzimidazolyl, indazolyl, benzoxathiolyl, benzodioxolyl,benzodithiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl,furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl,thienopyridyl, benzothienyl, cyclopentaoxazinyl, cyclopentafuranyl,benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl,isoquinolinyl, benzopyranyl, pyridopyridazinyl and pyridopyrimidinyl.

For the purposes of the present application, the term “animal” is meantto signify human beings, primates, domestic animals (such as horses,cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.) and othermammals. Generally, this term is used to indicate living creatureshaving highly developed vascular systems.

For the purposes of the present invention, agonists or agents or ligandsare molecules or compounds that bind to and modulate the function of thenicotinic receptor. Preferred agents are receptor-specific and do notcross the blood-brain barrier, such as DMPP. Useful agents may be foundwithin numerous chemical classes, though typically they are organiccompounds and preferably; small organic compounds. Small organiccompounds have a molecular weight of more than 150 yet less than about4,500, preferably less than about 1500, more preferably, less than about500. Exemplary classes include peptides, saccharides, steroids,heterocyclics, polycyclics, substituted aromatic compounds, and thelike.

Nicotinic agonists would not necessarily replace all drugs that arecurrently used to specifically treat inflammatory lung diseases and theairflow obstruction that is often associated with these diseases.Bronchodilators remain useful for the immediate release ofbronchospasms. However, bronchodilators have no effect on the underlyingcause of inflammation.

Nicotinic agonists may be useful as a steroid sparing or replacing drug.By targeting their delivery to the lung phagocytes, these drugs could behelpful in controlling both airway and interstitial inflammation. Onemajor advantage of nicotinic agonists over corticosteroids, besidesbeing expected to have fewer side effects, is the fact that theseagonists may have a direct effect on fibroblasts and could thereforeprevent or reverse fibrosis in the airways and in the lungs, somethingcorticosteroids cannot do. Interstitial fibrosis is the hallmark if IPF,a major sequel of HP and sarcoidosis, and airway fibrosis is aprevailing finding in chronic asthma (57).

Other substances are actively being studied as potential new treatmentsfor inflammatory lung diseases. Many cytokines are specifically targeted(e.g. IL-5, IL-13, IL-16 and the like) (62). It is believed that becauseof the complexity of pathways involved in inflammation, any one specificcytokine or other inflammatory mediator is unlikely to have asignificant impact on the treatment of these lung diseases. Nicotinicreceptor agonists as well as analogs and derivatives thereof, not unlikecorticosteroids, have the advantage of targeting a broad spectrum of theinflammatory response. Therein lies their potential in the treatment ofinflammatory lung diseases.

Selected agents may be modified to enhance efficacy, stability,pharmaceutical compatibility, and the like. Structural identification ofan agent may be used to identify, generate, or screen additional agents.For example, where peptide agents are identified, they may be modifiedin a variety of ways as described above, e.g. to enhance theirproteolytic stability. Other methods of stabilization may includeencapsulation, for example, in liposomes, etc. The subject bindingagents are prepared in any convenient way known to those skilled in theart.

For therapeutic uses, agents affecting nicotinic receptor function maybe administered by any convenient means. Small organics are preferablyadministered orally; other compositions and agents are preferablyadministered parenterally, conveniently in a pharmaceutically orphysiologically acceptable carrier, e.g., phosphate buffered saline, orthe like. Typically, the compositions are added to a retainedphysiological fluid such as blood or synovial fluid.

In accordance with the invention, there is provided another embodimentwhich is a pharmaceutical composition for treating pulmonaryinflammatory diseases comprising a nicotinic receptor agonist and apharmaceutically acceptable excipient.

The carrier(s) or excipient(s) must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notbeing deleterious to the recipient thereof.

In an alternative embodiment, there is provided a pharmaceuticalcomposition for treating pulmonary inflammatory diseases comprising

i) a compound of formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N,Ya is one or more optional substituent selected from halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio, of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, aralkyl, aryl of 6 to 10 carbon atomsand 3 to 10 membered heterocyclen is an integer from 0 to 2,J is a counter ion;or ii) a compound having the formula:

wherein R₃ is selected from

Xb is N or N⁺—R₁₀,R₄ is one or more substituent selected from hydrogen, halogen, amino,amidino, amido, azido, cyano, guanido, hydroxyl, nitro, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 10 carbon atoms, alkoxy of 1 to 10 carbon atoms,alkylthio of 1 to 10 carbon atoms, alkylamino of 1 to 10 carbon atoms,alkanol of 1 to 10 carbon atoms, aralkyl, aryl of 6 to 10 carbon atoms;each of R₁₀, R₁₁ and R₁₂ are independently alkyl of 1 to 10 carbonatoms,provided that a counterion is present when Xb is N⁺—R₁₀;or iii) a compound having the formula:

wherein Xc is NR₁₃ or N⁺—R₁₃R₁₄, wherein R₁₃ and R₁₄ are independentlyalkyl of 1 to 10 carbon atomsR₅ is a 3 to 10 membered heterocycle,provided that a counterion is present when Xc is N⁺—R₁₃R₁₄;or iv) a compound having the formula:

wherein W is O or S;each of Yc and Yd are independently a substituent selected fromhydrogen, halogen, amino, amidino, amido, azido, cyano, guanido,hydroxyl, nitro, nitroso, urea, sulfate, sulfite, sulfonate,sulphonamide, phosphate, phosphonate, acyl, acyloxy, alkyl of 1 to 10carbon atoms, alkoxy of 1 to 10 carbon atoms, alkylthio of 1 to 10carbon atoms, alkylamino of 1 to 10 carbon atoms, alkanol of 1 to 10carbon atoms, aralkyl, aryl of 6 to 10 carbon atoms;wherein Xd is NR₁₅ or N⁺—R₁₅R₁₆, wherein R₁₅ and R₁₆ are independentlyalkyl of 1 to 10 carbon atoms;provided that a counterion is present when Xd is N⁺—R₁₅R₁₆;anda pharmaceutically acceptable excipient.

In one embodiment, the pharmaceutical composition as defined herein maybe further comprising one or more therapeutic agent selected from abronchodilating agent, an anti-inflammatory agent, a leukotrienereceptor antagonist or a phosdiesterase inhibitor (PDE) such as PDE IV,

In a further embodiment, the bronchodilating agent is β2 agonists oranticholinergics.

In a further embodiment, the an anti-inflammatory agent iscorticosteroids.

In a further embodiment, the PDE inhibitor is PDE IV.

In another embodiment, the present invention provides a combinationcomprising a therapeutically effective amount of a compound useful inthe method of the present invention, and a therapeutically effectiveamount of at least one or more therapeutic agent.

It will be clear to a person of ordinary skill that if a furtheradditional therapeutic agent is required or desired, ratios will bereadily adjusted. It will be understood that the scope of combinationsdescribed herein is not limited to the therapeutic agents listed herein,but includes in principles any therapeutic agent useful for theprevention and treatment of pulmonary inflammatory diseases.

For peptide agents, the concentration will generally be in the range ofabout 50 to 500 μg/ml. Alternatively, it may administered in anacceptable range of from about 1 mg to a few 10 g or more per Kg in abody weight basis) in the dose administered. Other additives may beincluded, such as stabilizers, bactericides; etc. These additives willbe present in conventional amounts.

It will be appreciated that the amount of a compound of the inventionrequired for use in treatment will vary not only with the particularcompound selected but also with the route of administration, the natureof the condition for which treatment is required and the age andcondition of the patient and will be ultimately at the discretion of theattendant physician or veterinarian. Generally, the amount administeredwill be empirically determined, typically in the range of about 10 μg to1000 mg/kg of the recipient or 10 μg to 100 mg/kg or 10 μg to 1 mg/kgfor example.

The desired dose may conveniently be presented in a single dose or asdivided dose administered at appropriate intervals, for example as two,three, four or more doses per day.

While it is possible that, for use in therapy, a compound or combinationof the invention may be administered as the raw chemical it ispreferable to present the active ingredient as a pharmaceuticalcomposition.

As examples, many such therapeutics are amenable to direct injection orinfusion, topical, intratracheal/nasal administration e.g. throughaerosol, intraocularly, or within/On implants (such as collagen, osmoticpumps, grafts comprising appropriately transformed cells, etc. withtherapeutic peptides.

Pharmaceutical compositions also include those suitable for oral, nasal,topical (including buccal and sub-lingual), transdermal, or parenteral(including intramuscular, sub-cutaneous and intravenous) administrationor in a form suitable for administration by inhalation. The formulationsmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association theactive compound with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation.

Pharmaceutical compositions suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of, for example, aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The compounds and combinations according to the invention may also beformulated for parenteral administration (e.g. by injection, for examplebolus injection or continuous infusion) and may be presented in unitdose form in ampoules, pre-filled syringes, small volume infusion or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing an/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

Compositions suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

For administration by inhalation the compounds and combinationsaccording to the invention are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation, the compounds andcombinations according to the invention may take the form of a drypowder composition, for example a powder mix of the compound and asuitable powder base such as lactose or starch. The powder compositionmay be presented in unit dosage form in, for example, capsules orcartridges or e.g. gelatin or blister packs from which the powder may beadministered with the aid of an inhalator or insufflator.

Two animal models were used to study the effects of nicotinicantagonists in inflammatory pulmonary diseases; an HP model and anasthma model. With both of these models, the effects of nicotinicreceptor agonists (both selective and non-selective) were studied onlung physiology, and inflammation. In vitro studies were performed usingisolated inflammatory cells from the animal studies or from patients aswell as commercially available cell lines in an attempt to understandthe mechanisms by which nicotinic agonists down-regulate inflammation.

Initially, experiments were conducted with non-specific agonists, i.eagonists that bind to all nicotinic receptor subunits (nicotine,dimethylphenylpiperazinium (DMPP) and epibatidine) (13, 42). A β4subunit specific agonist, cytisine (42), was also tested to see whethera specific stimulation could also have anti-inflammatory effects.

Example I In Vivo HP Studies—I-Hypersensitivity-Like Inflammation

Effect of nicotinic agonists on long term-induced hypersensitivitypneumonitis (HP) In mice.

It is shown that the stimulation of nicotinic receptors with nicotinedown-regulates the immune response to HP antigens via inflammatorycytokine Suppression and inhibition of specific antigen-mediated,cellular activation.

This model was selected because, as mentioned previously, the incidenceof HP is lower in smokers than in non-smokers (50), and because thismodel is well described. HP was induced by the administration ofSaccharopolyspora rectivirgula (SR) antigen, the causative agent offarmer's lung (51), a form of HP. Mice were simultaneously treated withintra-peritoneal (IP) nicotine, with doses ranging from 0.5 to 2.0mg/kg, twice a day. Nicotine administration significantly reduced thenumber of total cells found in the bronchoalveolar lavage (BAL) of thesemice. The population that was the most affected by nicotine treatmentwere lymphocytes as seen in FIG. 1. It will be seen that there was amarked inhibition of total cell counts in nicotine treated mice duemainly to a decrease in the lymphocyte population. Pulmonary macrophagesand lymphocytes were isolated, and stimulated with anti-CD3+recombinantIL-2. The production of IFN-γ mRNA by these cells, a cytokine known tobe involved in the development of HP and other pulmonary inflammatorydiseases (52), was measured. Cells from nicotine treated animals showedsignificantly lower expression of IFN-γ mRNA than cells from non-treatedanimals. FIG. 2 illustrates that a significant inhibition of IFN-γ mRNAwas observed.

Example II In Vitro Studies Showing the Effect of Nicotinic Agonists onCytokine Expression

To further clarify the mechanisms involved in suppressive effect ofnicotine in the in vivo model, an alveolar macrophage cell line wasused.

The effect of nicotine or DMPP treatment on AMJ2-C11 cells was tested onTNF-α, IL-10 mRNA expression by RT-PCR. These cytokines are involved inthe development of pulmonary inflammatory diseases such as HP, asthmaand sarcoidosis (52-55). Nicotine and DMPP treatments showed a greatdecrease in TNF mRNA expression (up to a 98% reduction of expression inLPS stimulated cells treated with 40 μM nicotine), but not in adose-dependent manner. Reference is made to FIG. 3 where results areexpressed as a % of expression, 100% being attributed to the LPS alonegroup. The intensity of the band was obtained by dividing the intensityof the TNF-α band by that of β-actin. Treatment of stimulated cells withdifferent doses (40 to 160 μM for nicotine and DMPP) induced a drop ofTNF-α mRNA expression. The greatest effect was obtained with the 40 μMconcentration of nicotine (a 98% reduction of expression), while alldoses of DMPP caused a 60 to 50% reduction of expression. Similarresults were observed with SR-stimulated cells. Reference is made toFIG. 4 where results are expressed as described in FIG. 5. Treatment ofstimulated cells with different doses (80 and 160 μM for nicotine and 40to 160 μM for DMPP) induced a down-regulation of TNF-α mRNA expression,Only the 160 μM dose of nicotine had an effect on mRNA expression, whilethe 40 and 80 μM doses of DMPP induced up to 60% of reduction of TNF-αmRNA expression. This non-dose dependent response can be explained bynicotinic receptor desensitization due to a large quantity of agonist inthe medium. IL-10 mRNA expression was also reduced by nicotine and DMPPtreatment. The maximal down-regulation occurred at a dosage of 40 μMnicotine (LPS stimulated; 88% reduction of mRNA expression; reference ismade to FIG. 5 where results are expressed. Treatment of stimulatedcells with different doses (40 to 160 μM for both nicotine and DMPP)induced a down-regulation of IL-10 mRNA expression. The largest drop ofexpression (a 87% reduction) occurred with 40 μM nicotine. DMPP induceda 55 to 40% reduction of expression for all three doses. At a dosage of80 μM DMPP a 87% IL-10 mRNA expression reduction is observed inSR-stimulated cells, the results are given in FIG. 6. Treatment ofSR-stimulated cells with different doses (80 and 160 μM for nicotine and40 to 80 μM for DMPP) induced a down-regulation of IL-10 mRNAexpression. The greatest drop in mRNA expression with the nicotinetreatment occurred at 160 μM (60% drop of expression), and at 80 μM (90%drop of expression) with the DMPP treatment.

Another macrophage cell line (RAW 264.7; ATCC) was used to test theeffect of DMPP on IFN-γ expression by RT-PCR, because AMJ2-C11 cells didnot appear to express IFN-γ mRNA (data not shown). Cells were stimulatedwith 50 μg/ml of SR antigen and incubated with DMPP at doses rangingfrom 40 to 160 μM. DMPP treatment reduced the expression of IFN-γ inthese cells by up to 75% with the 40 μM dose, Reference is made to FIG.7 where results are expressed as described in FIG. 5. Treatment ofstimulated cells with different doses of DMPP induced a reduction inIFN-γ mRNA expression. The largest drop of expression (a 80% reduction)occurred with 40 μM DMPP.

Example III In Vitro Effects of Nicotinic Agonists on Co-StimulatoryMolecule Expression

The effects of nicotine and DMPP on B7 (CD80) molecule expression weretested in vitro. AMJ2-C11 cells (mouse alveolar macrophages, from theATCC) were incubated with 40 μM nicotine or DMPP and stimulated with LPS(0.1 μg/ml) or SR antigen (50 μg/ml) for 48 hours. The percentage ofexpression of CD80 in treated cells was about one half of the expressionfound in LPS and SR stimulated non-treated cells. Reference is made toFIG. 8 (a) which shows that nicotine treatment (40 μM for 48 h) reducedthe expression to 20% in LPS stimulated cells. Reference is also made toFIG. 8 (b) which shows that DMPP treatment (40 μM for 48 h) reduced theexpression to 17% in LPS stimulated cells and 20% in SR stimulatedcells.

Example IV Studies on Human BAL Cells (AM and Lymphocytes)

Since one goal was to treat patients with DMPP or similar drugs, theeffect of this drug was verified on lymphocytes from patients with HP.BAL were performed on patients with HP. Lymphocytes were isolated fromthe other BAL cells, stimulated with PHA and incubated with DMPP. Thedose-response of DMPP were tested on cytokine mRNA production (byRT-PCR) for IFN-γ. Reference is made to FIG. 9 which shows that DMPPtreatment reduced expression of IFN-γ in these cells.

A broncho-alveolar lavage was performed on a normal patient, andalveolar macrophages were isolated. SR-stimulated and nicotine or DMPPtreated cells showed once again about half of the expression of CD86than non-treated cells. Reference is made to FIG. 10 which shows thatcells that were treated with DMPP express 50% less CD86 than non-treatedcells.

Example V Investigation of the Effect of Other Nicotinic Agonists on theShort Term SR-Induced Acute Inflammation

The Intranasal instillation of Saccharopolyspora rectivirgula (SR)antigens, the causative agent for farmer's lung, to mice, induces aprominent inflammatory response in the lung. Neutrophils are the firstinflammatory cells recruited at the site of inflammation. Treatment ofmice with DMPP (0.5 mg/kg), nicotine (0.5 mg/kg) and epibatidine (2μg/kg) had a marked inhibitory effect on SR-induced inflammation.Reference is made to FIG. 11 which shows that treatment with nicotineand epibatidine had a significant inhibitory effect on SR-inducedinflammation after 24 hours, Nicotinic agonists were administeredintra-nasally in 50 μl volume every 6 h and mice were sacrificed 24 hrafter SR instillation.

A significant inhibitory effect was observed with nicotine andepibatidine but not with DMPP. However, after increasing the number ofmice treated or not treated with DMPP to 15, we did observe asignificant inhibition compared to the non-treated group (FIG. 12).

Levels of TNF (a pro-inflammatory cytokine) are lower in thebroncho-alveolar lavage of DMPP-treated mice (FIG. 13 shows that DMPPdecreased significantly BALF TNF levels) indicating that thedown-regulation of inflammation may result from lower TNFconcentrations.

Example VI In Vivo Asthma Model

Similar experiments were performed in ovalbumin-sensitized mice. DMPPallegedly decreases both the inflammatory response and thehyper-responsiveness to inhaled allergens and methacholine.

Groups of Balb/c mice were sensitized by intra-peritoneal injection of20 μg OVA protein (chicken egg albumin; Sigma-Aldrich) emulsified in 2mg aluminum hydroxide in PBS. After 4 weeks, challenge doses of 1.5%/50μl OVA were administered intranasally. The challenge was performed dailyfor 3 consecutive days and then the mice assessed for allergicinflammation of the lungs 24 h after the last aerosol exposure. Groupsof mice were treated with various concentrations of DMPP during thechallenge period. Broncho-alveolar lavage (BAL) was performed and thefluid centrifuged at 400 g to separate cells from liquid. FIG. 14 showsthat The number of cells was highly elevated in OVA challenged andnon-treated mice. The DMPP treatment significantly reduced cell countsat the 0.5 and 2.0 mg/kg doses. FIG. 15 shows that the OVA challengedmice (OVA OVA) had more eosinophils and lymphocytes in their BALcompared to the control group (sal sal). The DMPP treatmentsignificantly reduced the presence of both eosinophils and lymphocytesin BAL In all groups (n=8; p<0.05). FIG. 16 shows that the OVAchallenged mice (OVA OVA) had more eosinophils and lymphocytes in theirBAL compared to the control group (sal sal). The DMPP treatmentsignificantly reduced the presence of both eosinophils and lymphocytesin BAL in all groups (n=8; p<0.05). FIG. 17 shows that The DMPPtreatment significantly reduced eosinophil and lymphocyte counts in the0.1 and 0.5 mg/kg doses, 0.5 mg/kg being the most effective dose for theanti-inflammatory effect of DMPP.

The supernatants were used to determine lung IL-5 levels. The total,number of BAL cells and differential cell counts were evaluated. FIG. 18shows that the OVA challenges increased IL-5 levels in BAL, while theDMPP treatment had a significant inhibitory effect on IL-5 levels in the0.5 mg/kg treated-group of mice.

The experiment was repeated with the optimal dose of DMPP to assess theairway responsiveness.

Measurement of AHR

Airway hyper-reactivity (AHR) in response to metacholine was measured inanesthetized, tracheotomized, ventilated mice using acomputer-controlled ventilator (FlexiVENT™).

Increasing doses of metacholine (0 mg/kg-32.5 mg/kg) were administeredthrough the jugular vein. FIG. 19 shows that DMPP-reduced the % ofaugmentation of lung resistance compared to asthmatic mice. FIG. 20shows that DMPP significantly reduced the PC200 in treated-mice comparedto asthmatic mice (p=0.04; n=6).

Example VII Effect of Agonist Treatment on mRNA Expression of IL-4

The effect of agonist treatment on mRNA expression of IL-4, a cytokinethat is well known to be involved in the development of asthma, was alsotested (53). Nicotine decreased IL-4 mRNA expression by up to 92% with40 μM (FIG. 9) DMPP completely blocked IL-4 mRNA expression. Referenceis made to FIG. 21 which shows results expressed as described in FIG. 5.Cells were treated with different doses (40 to 160 μM for both nicotineand DMPP). The nicotine treatment induced a drop in the IL-4 mRNAexpression (up to a 90% reduction of expression in the 40 μM group). Asdemonstrated previously, there was no IL-4 mRNA expression when cellswere stimulated with SR antigen.

Example VIII Action of Various Agonists on Eosinophil Transmigration

To further investigate the down-regulatory effect of nicotinic agonistson inflammation in asthma, we tested the action of various agonists oneosinophil transmigration.

Infiltration of eosinophils and other inflammatory cells into lungtissues is an important feature of asthma and the cause of airwayinflammation and hyper-responsiveness. The passage of inflammatory cellsfrom the circulation to the lung involves migration through the vascularendothelium, the basement membrane, and extra-cellular matrixcomponents. Inflammatory cells cross the basement membrane by producingproteinases. In these preliminary in vitro experiments, the effects ofvarious nicotinic agonists on the migration of purified bloodeosinophils through an artificial basement membrane (Matrigel® coatedchemotaxis chamber) were investigated. DMPP induced a dose-relatedinhibition of eosinophils transmigration (FIG. 22 shows that DMPPinduces a dose-related inhibition of eosinophil transmigration across anartificial basement membrane), while this effect was reversed by theantagonist mecamylamine (MEC) (FIG. 23 shows that mecamylamine reversesthe effect of DMPP, suggesting that nicotinic receptor activation isnecessary for the DMPP inhibitory effect). This inhibitory effect isfurther confirmed with other nicotinic agonists including nicotine,epibatidine and cytisine (FIG. 24) that all reduce blood eosinophiltransmigration. Results are expressed as a percentage of inhibition(agonists-treated cells) compared to the control condition without theagonists.

These results suggest that nicotinic agonists down-regulate thesynthesis or activation of proteinases that degrade basement membranecomponents, thus inhibiting the migration of eosinophils into lungmucosa.

Example IX Effect of Nicotinic Agonists on Collagen Production

Asthma is characterized by airway structural changes, includingsub-epithelial collagen deposition, that may be a cause for thechronicity of the disease. An imbalance between collagen synthesis andits degradation by fibroblasts may be involved in this process (56). Inpreliminary experiments, we investigated the effects of nicotinicagonists on collagen A1 synthesis produced by primary normalfibroblasts. Collagen A1 gene expression was evaluated by RT-PCR.

The results are expressed percentage gene expression in agonists treatedcells compared to non-treated cells.

DMPP inhibits collagen A1 gene expression in a dose-dependent manner(FIG. 25). Nicotine has a slight inhibitory effect at 1 and 10 μM,whereas higher concentrations had no effects (FIG. 26), probably due toa desensitization of the receptors. Lower doses may be necessary toachieve an inhibition and will be tested. The inhibitory effect is alsoobserved with epibatidine (FIG. 27).

Similar tests were carried out with the following analogues of DMPP andequivalent results were obtained.

Example X Effects of DMPP Analogs

Based on our DMPP results, four (4) new DMPP analogs were developed andtested for their anti-inflammatory effects, improvedhyper-responsiveness properties and smooth muscle-relaxing effects.Similarly to DMPP, ASM-002, ASM-003, ASM-004 and ASM-005 are syntheticagonists of nicotinic acethylcholine receptors. They are highlyhydrophilic due to their quaternary salt structure, and therefore arenot likely to cross easily the blood-brain barrier. Consequently theyare less likely to induce addiction.

Example XI Anti-Inflammatory Effects

Effect of DMPP Analogs on Tumor Necrosis Factor (TNF) Release

Human monocytes were isolated from the blood of asthmatic patients byFicoll-paque density gradient, let to adhere to tissue culture platesand stimulated with LPS (100 ng/ml) for 18 hours at 37° C. with orwithout increasing concentrations of nicotine. The release of TNF, apotent pro-inflammatory mediator, was measured in the cell culturesupernatant by the ELISA method. Results are expressed as a percentagerelease from LPS-stimulated untreated cells (FIG. 28). Except forASM-005, all analogs tested had an inhibitory effect on TNF release (n=8to 10; p from 0.01 to 0.007).

Example XII Effect of DMPP Analogs on Leukotriene C4 (LTC4) Production

Blood eosinophils, the most increased inflammatory cells in asthma, wereisolated by negative selection using bead-conjugated anti-CD16monoclonal antibody and magnetic activating cell sorting. Cells werepre-Incubated for 18 hours with the various DMPP analogs and thenstimulated with 1 □M platelet-activating factor (PAF) to produce LTC4which was measured by enzyme immunoassay.

The results indicate that 3 out of 4 analogs tested are able todown-regulate LTC4 release (Table 1).

TABLE 1 Effects of DMPP and analogs on LTC4 release. LTC4 pg/ml —1725.80 DMPP 545.00 ASM002 246.40 ASM003 613.90 ASM004 601.60

Example XIII Smooth Muscle Relaxing Effects

Effect of DMPP Analogs on Mouse Tracheal Airway Smooth MuscleResponsiveness

To demonstrate the relaxing effects of DMPP analogs on airway smoothmuscle cells, isometric studies were performed on isolated mousetracheas. Tracheal rings were mounted isometrically to force transducersin organ baths containing Krebs bicarbonate solution at 37° C. andbubbled with 95% O₂-5% CO₂, pre-contracted with submaximal concentrationof metacholine (10⁻⁵) and cumulative doses of the analogs were added tothe baths. Changes of tension are recorded. Results are expressed as apercentage of maximal contraction (FIG. 29).

Similarly to DMPP, its analogs induced a dose dependant relaxation oftracheal smooth muscles pre-contracted with metacholine.

Overall these results indicated that ASM-002, ASM-003, ASM-004 andASM-005 the new synthesized analogs presented similar anti-inflammatoryand smooth-muscle relaxing effects as DMPP.

Example XIV Mouse Model

Effects of ASM-002 on Lung Inflammation

Ovalbumin-sensitized mice (n=8) were challenged with the allergen andsimultaneously treated intra-nasally with increasing concentrations ofASM-002 (0.5 to 4 mg/kg/d) for 3 days. The number of cells recovered bybroncho-alveolar lavage was used as a measure of lung inflammation.

As shown in FIG. 30, ASM-002 significantly inhibits in a dose dependantmanner the cellular inflammation in the lungs of asthmatic mice(ED₅₀=0.71 mg/kg, n′=8).

Example XV Mouse Model

Effects of ASM-002 on Lung Resistance in a Mouse Model of Asthma

Lung response to a broncho-constrictive agent, metacholine, was measuredby a Flexi-vent® apparatus. Ovalbumin-sensitized animals were treatedintra-nasally with ASM-002 (4 mg/kg) during 3 days and 10 minutes priorto the metacholine challenge and compared to untreated OVA-sensitizedanimals. A negative control group of un-sensitized animals and apositive control group that received Salbutamol (Ventolin) 10 minutesbefore the metacholine challenge were also included.

The results show (FIG. 31) an increase in lung resistance, induced bymetacholine, in OVA-sensitized mice compared to the negative controlgroup. A significant reduction (return to baseline levels) in lungresistance is observed in ASM-002-treated mice compared to untreatedmice (n=8, p<0.02). This effect is similar to that obtained withSalbutamol (Ventolin™), the most common brochodilator currently used inasthma to relieve broncho-constriction symptoms (n=4, p<0.02).

Example XVI Dog Asthma Model

In this model 12 dogs naturally sensitized to the roundworm Ascaris suumwere used in a cross-over study design. Four groups of 3 dogs wererandomly formed, exposed to the allergen, and each animal was lefteither untreated or received alternatively, ASM-002 (4 mg/kg 2× day inthe food), or prednisone (1 mg/kg 1× day in the food), the most commonlyused corticosteroid drug used to treat inflammation in asthma.

Comparative Effects of ASM-002 and Prednisone™ on Lung Inflammation

Cellular inflammation was evaluated in the bronchoalveolar lavages.

As shown in FIG. 32, ASM-002 (8 mg/kg) inhibits significantly thecellular inflammation in the lungs of asthmatic dogs with a similarefficacy as Prednisone™, the most frequently used anti-inflammatory drug(n=12, p<0.05).

Example XVII Effects of ASM-002 in a Dog Model of LungHyper-Responsiveness

Hyper-responsiveness is described as the capacity of the lung to react(to increase lung resistance) to a non-specific external stimuli likemetacholine or to allergens A hyper-responsive allergen-sensitized dog(asthmatic) will react to lower concentrations of metacholine comparedto a non allergic dog. Similarly, improvement in lunghyper-responsiveness is shown by an increase in metacholineconcentrations necessary to induce the same level of lung resistance.

Increasing concentrations of metacholine were administered with orwithout treatment with ASM-002 or Prednisone™ and lung resistancerecorded.

As shown in FIG. 33, ASM-002 decreased lung resistance in 7 out of 12hyperresponsive dogs. None of the 12 dogs showed improvedhyper-responsiveness with prednisone (p=0.005).

Example XVIII Muscle-Relaxing Properties of ASM-002

To further demonstrate the relaxing effects of ASM-002 on airway smoothmuscle cells, isometric studies were performed on isolated mousetracheas, bronchial rings from dog's lungs and bronchial rings fromresected human lungs. As described previously, tracheal or bronchialrings were mounted isometrically to force transducers in organ bathscontaining Krebs bicarbonate solution at 37° C. and bubbled with 95%O₂-5% CO₂, pre-contracted with submaximal concentration of metacholinecumulative doses of ASM-002 added. Changes of tension are recorded.Results are expressed as a percentage of maximal contraction for mouse(FIG. 34, p=0.002), dog (FIG. 35, p=0.004) and human (FIG. 36)

These results of examples XIV to XVIII showed that ASM-002 present arelaxing effect on pre-contracted mice tracheas, dog and human bronchi.

Example XX In Vitro Studies

The anti-inflammatory activity of ASM-002 was observed in vivo in miceand dogs in previous Examples. To further characterize this effect, thedrug was tested for its capacity to inhibit the release of 2 potentinflammatory mediators by human blood cells Isolated from asthmaticpatients

Tumor necrosis factor (TNF) is a mediator released in inflammatorystates. Human blood monocytes were stimulated in vitro withlipopolysaccharide (LPS) to produce large amounts of TNF, increasingdoses of ASM-002 were added and the levels of TNF were measured (FIG.37, EC₅₀=3 μM, n=6, p=0.0045 at 5 μm, 0.0014 at 10 μm and 0.0003 at 50μm). A dose-dependant inhibition of TNF release was observed withASM-002

Example XXI Comparative Effects of ASM-002 with DMPP and Dexamethasoneon TNF Production by LPS-Stimulated Blood Monocytes

As shown in FIG. 38, results are expressed as a percentage fromuntreated control cells, all drugs were added at a 40 μM concentrationand are the mean of 5 different experiments (5 subjects). ASM-002inhibits TNF release from human blood monocytes as well as dexamethasoneand DMPP (p=from 0.02 to 0.001)

Leukotriene C₄ (LTC₄) is a inflammatory lipid mediator highly producedin asthma, it is released in large amounts by blood eosinophils.

Human blood eosinophils were isolated from blood of asthmatic patients,stimulated in vitro with platelet activating factor (PAF) to producelarge amounts of LTC4, and treated or not with 80 μM ASM-002.

A significant inhibition of LTC4 production by ASM-treated eosinophilswas observed (FIG. 39, p=0.0007). The results represent an average of 6different experiments (6 patients).

The results showed that ASM-002 present combined anti-inflammatory andbroncho-dilating properties and improved hyperresponsiveness, whichcould be highly effective for the relief and treatment of asthma andother obstructive respiratory diseases.

Example XXII Other Nicotinic Acetylcholine Receptor Analogs

Other analogs such as nicotine, cytisine and epibatidine as describedherein can be used as nicotinic receptors inhibitors in the treatment ofpulmonary inflammation.

Anti-Inflammatory Effects:

Human blood monocytes were isolated by Ficoll-paque density gradient,let to adhere to tissue culture plates and stimulated with LPS (100ng/ml) for 18 hours at 37° C. with or without increasing concentrationsof nicotine analogues. The results obtained are disclosed in FIG. 40,significance levels are shown in Table 2.

TABLE 2 Significance levels of the effects of Nicotinic analogs on LPSstimulation. p = Concen- tration Nicotine ASM-N1 ASM-N2 ASM-N3 ASM-N4(M) p = p = p = p = p = 10⁻⁴ 0.034 0.011 0.006 0.037 0.035 10⁻⁵ 0.0320.015 0.001 0.008 0.039

A significant decrease of TNF release was observed with increasingconcentrations of the four nicotine analogs.

Example XXIII

1-Phenylpiperazine (1 eq), iodoethane (1 eq), and sodium carbonate (2eq) were mixed in tert-butanol. The mixture was refluxed for 20 hours.The mixture is then dissolved in chloroform and extracted with waterthree times. The organic layer was washed with 1N aqueous HCl solutionthree times. The aqueous layer was then basified to a basic pH with NaOHpellets. The basic aqueous layer was then extracted with chloroformthree times and the combined organic extracts dried over Na₂SO₄ andevaporated to dryness. The crude product was purified using silica gelflash chromatography using a gradient of 0-5% MeOH in chloroform. Thedesired product was obtained as a yellow oil. (yield. 52%).

N-ethylphenylpiperazine (1 eq, 0.6 mmol) and iodomethane (excess >10 eq,1 ml) were stirred in ether at room temperature for 4 days. Theresulting white precipitate of ASM-003 was isolate by vacuum filtration.(yield 75%).

Example XXIV

1-Phenylpiperazine (1 eq), iodopropane (1 eq), and sodium carbonate (2eq) were mixed in tert-butanol. The mixture was refluxed for 20 hours.The mixture is then dissolved in chloroform and extracted with waterthree times. The organic layer was washed with 1N aqueous HCl solutionthree times. The aqueous layer was then basified to a basic pH with NaOHpellets. The basic aqueous layer was then extracted with chloroformthree times and the combined organic extracts dried and evaporated todryness. The crude product was purified using silica gel flashchromatography using a gradient of 0-5% MeOH in chloroform. The desiredproduct was obtained as a yellow oil. (yield. 83%).

N-propylphenylpiperazine (1 eq, 0.6 mmol) and iodomethane (excess >10eq, 1 ml) were mixed and stirred at room temperature in ether for 2days. The mixture was then refluxed for 48 hours with an additionalamount of iodomethane (>10 eq) with a (1:1) mixture of THF and ether.The mixture was evaporated and diluted in ether to yield a whiteprecipitate of ASM-004 isolated by vacuum filtration. (yield 86%).

Example XXV

N-ethylphenylpiperazine prepared in example XXIII (1 eq, 0.5 mmol) andiodoethane (excess >10 eq, 1 ml) were stirred in ether at roomtemperature for 2 days. The mixture was then refluxed for 48 hours withan additional amount of iodoethane (>10 eq), with a (1:1) mixture of THFand ether. The mixture was evaporated and diluted in ether to yield awhite precipitate of ASM-005 isolated by vacuum filtration (yield 62%).

or

N-ethylphenylpiperazine (1 eq, 3.94 mmol) and iodoethane (excess >10 eq,3 ml) were stirred in acetonitrile at room temperature The mixture Wasevaporated and diluted in ether to yield a white precipitate of ASM-005Isolated by vacuum filtration (yield 27%).

Example XXVI

N-propylphenylpiperazine (1 eq, 0.51 mmol) and iodoethane (excess >10eq, 1 ml) were stirred in ether at room temperature for 2 days Themixture was then refluxed for 48 hours with an additional amount ofiodoethane (>10 eq), with a (1:1) mixture of THF and ether. The mixturewas evaporated and diluted in ether to yield a white precipitateisolated by vacuum filtration (yield 11%).

or

N-propylphenylpiperazine eq, 0.1 mmol) et l'iodoethane (excess >10 eq, 1ml) were stirred in refluxing acetone for 24 hours. The mixture wasevaporated and diluted in ether to yield a white precipitate isolated byvacuum filtration (yield 75%).

Example XXVII

N-propylphenylpiperazine (1 eq, 0.53 mmol) and iodopropane (excess >10eq, 1 ml) were stirred in ether at room temperature for 2 days Themixture was then refluxed for 48 hours with an additional amount ofiodopropane (>10 eq, 1 ml), with a (1:1) mixture of THF and ether. Themixture was evaporated and diluted in ether to yield a white precipitateisolated by vacuum filtration (yield 10%).

Example XXVIII

In a flame-dried round bottom flask under nitrogen, iodobenzene (1 eq,1.47 mmol), N-methylhomopiperazine (1.2 eq, 1.76 mmol), ethylene glycol(2 eq, 2.94 mmol), CuI (5% mol) and K3PO4 (2 eq, 2.94 mmol) weresuspended in isopropanol (3 ml). The mixture was refluxed with stirringfor 17 hours. The resulting mixture was cooled down to room temperatureand water was added (5 ml). The mixture was extracted with ether (4×10ml) and the combined organic extracts washed with brine, dried overNa2SO4 and evaporated to dryness under vacuum. The crude product waspurified using silica gel flash chromatography using a gradient of 0% a7.5% (2M NH3)MeOH in chloroform. The desired product was obtained as ayellow oil. (yield 64%).

N-methylphenylhomopiperazine (1 eq, 0.36 mmol) and iodomethane(excess >10 eq, 1 ml) were stirred in ether at room temperature for 25hours. The mixture was evaporated under vacuum, diluted with ether andthe resulting white solid filtered under vacuum.1,1-dimethyl-4-phenylhomopiperazinium iodide (Yield: 66%), Meltingpoint: 158-160.

¹H NMR DMSO-d6 (ppm): (q, 2H) 7.18, (q, 2H) 6.74, (t, 1H) 6.64, (br s,2H, 3.74), (m, 2H) 3.52, (m, 2H) 3.44, (t, 2H) 3.40, (s, 6H) 3.17, (bs5, 2H) 2.21.

¹³C NMR DMSO-d6: 149, 129, 117, 112, 66, 65, 53, 47, 43, 22.

Example XXIX

Nicotine (160 mg, 0.987 mmol) was dissolved in diethylether (5 ml), anexcess of iodomethane (33 eq, 2 ml) was added and stirred in dark atroom temperature over night for 15 hours.

The mixture was filtered under vacuum and the solid washed withdiethylether. A white precipitate of ASM-N1 was obtained (yield 91%).

¹H NMR acetone-d6 (ppm): (m, 1H) 1.75, (m, 1H) 1.85, (m, 1H) 2.0, (s,3H) 2.26, (m, 2H) 2.42, (m, 1H) 3.25 (t, 1H) 3.59, (s, 3H) 4.67 (t, 1H)8.21, (d, 1H) 8.66, (d, 1H) 9.13, (s, 1H) 9.22.

Example XXX

To the previously obtained nicotine salt compound (ASM-N1) (100 mg, 0.32mmol) in anhydrous dichloromethane (15 ml) anhydre, was added an excessof iodomethane (33 eq, 0.64 ml) and stirred in dark at room temperatureover night for 18 hours.

The mixture was filtered under vacuum and the solid washed withdiethylether. A white precipitate was obtained (yield 26%).

¹H NMR acetone-d6 (ppm): (m, 3H) 2.26, (m, 1H) 2.71, (s, 3H) 2.82, (s,3H) 3.14 (m, 1H) 3.76, (m, 1H) 3.86, (s, 3H) 4.40, (t, 1H) 5.04, (t, 1H)8.31, (d, 1H), 8.85 (d, 1H), 9.17, (s, 1H) 9.31.

Example XXXI

Nicotine (390 mg, 2.4 mmol) was dissolved in diethylether (10 ml), anexcess of iodoethane (33 eq, 6.3 ml) was added and stirred in dark atroom temperature for 7 days.

The solvent was evaporated and dichloromethane was added (100 ml) tocause precipitation of a white-yellowish of ASM-N4.

The organic layer was evaporated and the resulting oil washed withdiethyl ether to yield ASM-N3.

¹H NMR acetone-d6 (ppm) of ASM-N3: (t, 3H) 1.70, (m, 1H) 1.82, (m, 1H)1.95, (s, 3H), 2.26 (m, 3H) 2.43, (m, 1H) 3.30 (m, 1H) 3.70, (q, 2H)4.95, (m, 1H) 8.20, (d, 1H) 8.69, (d, 1H) 9.29, (s, 1H) 9.39.

¹H NMR acetone-d6 (ppm) of ASM-N4: (2t, 3H) 1.2 et 1.5, (t, 3H) 1.75,(m, 1H) 1.85, (m, 2H) 2.05 (s, 3H) 2.41, (m, 1H) 2.71, (m, 2H) 3.45,(2q, 2H) 3.78 et 3.95, (m, 1H) 4.12, (q, 2H) 4.98, (m, 1H) 8.27, (d, 1H)8.86, (d, 1H) 9.40, (s, 1H) 9.66

Example XXXII

ASM-C1 was prepared using iodomethane (10 eq) in dichloromethane for 1hour in dark in a manner similar to what is described in example XXIX.The characterisation was consistent with the structure.

Example XXXIII

ASM-C2 was prepared using formaldehyde and formic acid in a similarmanner as that described in J. Med. Chem. (2001), 44, 3946-3955. Thecharacterisation was consistent with the structure.

Example XXXIV

ASM-C3 was prepared using iodomethane (40 eq) in dichloromethane for 20hours in the dark in a manner similar to what is described in exampleXXIX. The characterisation was consistent with the structure.

Example XXXV

ASM-C4 was prepared using ethylene oxyde in a similar manner as thatdescribed in II Farmaco 54 (1999) 438-451. The characterisation wasconsistent with the structure.

Example XXXVI

ASM-05 was prepared using iodomethane (40 eq) in dichloromethane for 18hours in the dark in a manner similar to what is described in exampleXXIX. The characterisation was consistent with the structure.

Example XXXVII

(+)-Epibatidine dihydrochloride was treated with triethyl amine (10 eq)in dichloromethane for 1 hour at room temperature and epibatidine wasthen isolated following standard isolation protocol.

ASM-E1 was prepared using epibatidine, formaldehyde and formic acid in asimilar manner as that described in J. Med. Chem. 2001, 44, 3946-3955The characterisation was consistent with the structure.

Example XXXVIII

ASM-E1 was prepared using iodomethane (40 eq) in dichloromethane for 17hours at room temperature in a manner similar to what is described inexample XXIX. The characterisation was consistent with the structure.

Although the present invention has been described herein above by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

REFERENCES

-   1, Cormier, Y. J., Belanger, and P. Durand. 1985. Factors    influencing the development of serum precipitins to farmer's lung    antigen in Quebec dairy farmers. Thorax 40(2):138-42.-   2, Cormier, Y., L. Gagnon, F. Berube-Genest, and M. Fournier. 1988,    Sequential bronchoalveolar lavage in experimental extrinsic allergic    alveolitis. The influence of cigarette smoking. Am Rev Respir Dis    137(5):1104-9.-   3. Cormier, Y., E. Israel-Assayag, G. Bedard, and C. Duchaine. 1998.    Hypersensitivity pneumonitis in peat moss processing plant workers.    Am J Respir Crit Care Med 158(2):412-7.-   4. Gariepy, L., Y. Cormier, M. Laviolette, and A. Tardif. 1989.    Predictive value of bronchoalveolar lavage cells and serum    precipitins in asymptomatic dairy farmers. Am Rev Respir Dis    140(5):1386-9.-   5. Lawrence, B. C., T. B. Fox, R. B. Teague, K. Bloom, and R. K.    Wilson. 1986. Cigarette smoking and bronchoalveolar T cell    populations in sarcoidosis. Ann N Y Acad Sci 465:657-64.-   6. Valeyre, D., P. Soler, C. Clerici, J. Pre, J. P. Battesti, R.    Georges, and A. S. Hance. 1988. Smoking and pulmonary sarcoidosis:    effect of cigarette smoking on prevalence, clinical manifestations,    alveolitis, and evolution of the disease. Thorax 43 (7):516-24,-   7. Rubin, D. T., and S. B. Hanauer, 2000, Smoking and inflammatory    bowel disease. Eur J Gastroenterol Hepatol 12(8):855-62.-   8, Thomas, G. A., J. Rhodes, J. T. Green, and C. Richardson, 2000.    Role of smoking in inflammatory bowel disease: implications for    therapy. Postgrad Med J 76(895):273-9.-   9, Guslandi, M. 1999. Nicotine treatment for ulcerative colitis. Br    J Clin Pharmacol 48(4):481-4.-   10. Guslandi, M. 1999. Long-term effects of a single course of    nicotine treatment in acute ulcerative colitis: remission    maintenance in a 12-month follow-up study. Int J Colorectal Dis    14(4-5):261-2.-   11. Rezvani, A. H., and E. D. Levin. 2001, Cognitive effects of    nicotine. Biol Psychiatry 49(3):258-67.-   12. Kelton, M, C., H. J. Kahn, C. L. Conrath, and P. A.    Newhouse. 2000. The effects of nicotine on Parkinson's disease.    Brain Cogn 43(1-3):274-82.-   13. Bertram, K. G. 1998. Basic and clinical pharmacology. Editions    Appelton and Lange. Stanford, Conn.-   14. Sekhon, H. S., Y. Jia, R. Raab, A. Kuryatov, J. F. Pankow, J. A.    Whitsett, S. Lindstrom, and E. R. Spindel. 1999. Prenatal nicotine    increases pulmonary alpha7 nicotinic receptor expression and alters    fetal lung development in monkeys. J Clin Invest 103(5):637-47.-   15. Maus, A. D., E. F. Pereira, P. I. Karachunski, R. M. Horton, D.    Navaneetham, K. Macklin, W. S. Cortes, E. X. Albuquerque, and B. M.    Conti-Flue, 1998. Human and rodent bronchial epithelial cells    express functional nicotinic acetylcholine receptors. Mol Pharmacol    54(5):779-88.-   16. Skiver, S. P., H. A. Bourdeau, C. T. Gubish, D. L. Tirpak, A. L.    Davis, J. D. Luketich, and S. M. Siegfried. 2000. Sex-specific    expression of gastrin-releasing peptide receptor: relationship to    smoking history and risk of lung cancer. J Natl Cancer Inst    92(1):24-33.-   17, Ferguson, D. G., M. A. Haxhiu, A. J. To, B. Erokwu, and I. A.    Dreshaj. 2000. The alpha3 subtype of the nicotinic acetylcholine    receptor is expressed in airway-related neurons of the nucleus    tractus solitarius, but is not essential for reflex    bronchoconstriction in ferrets. Neurosci Lett 287(2):141-5.-   18. Singh, S. P., R. Kalra, P. Puttfarcken, A. Kozak, S. Tesfaigzi,    and M. L. Sopori. 2000, Acute and chronic nicotine exposures    modulate the immune system through different pathways. Toxicol Appl    Pharmacol 164(1):65-72.-   19. Kalra, R., S. P. Singh, S. M. Savage, G. L. Finch, and M. L.    Sopori. 2000. Effects of cigarette smoke on immune response: chronic    exposure to cigarette smoke impairs antigen-mediated signaling in T    cells and depletes IP3-sensitive Ca(2+) stores. J Pharmacol Exp Ther    293(1):166-71.-   20. Sugano, N., K. Shimada, K. Ito, and S. Murai. 1998. Nicotine    inhibits the production of inflammatory mediators in U937 cells    through modulation of nuclear factor-kappaB activation. Biochem    Biophys Res Commun 252(1):25-8.-   21, Yates, S. L., M. Bencherif, E. N. Fluhler, and P. M.    Lippiello. 1995. Up-regulation of nicotinic acetylcholine receptors    following chronic exposure of rats to mainstream cigarette smoke or    alpha 4 beta 2 receptors to nicotine. Biochem Pharmacol    50(12):2001-8.-   22, Sopori, M. L., and W. Kozak. 1998. Immunomodulatory effects of    cigarette smoke. J Neuroimmunol 83(1-2):148-56.-   23. Lahmouzi, J, F. Simain-Sato, M. P. Defresne, M. C. De Palm, E.    Heinen, T. Grisar, J. J. Legros, and R. Legrand. 2000. Effect of    nicotine on rat gingival fibroblasts in vitro. Connect Tissue Res    41(1):69-80.-   24. Geng, Y., S. M. Savage, S. Razanai-Boroujerdi, and M. L.    Sopori. 1996. Effects of nicotine on the immune response. II.    Chronic nicotine treatment induces T cell anergy. J Immunol    156(7):2384-90.-   25. McCrea, K. A., J. E. Ensor, K. Nall, E. R. Bleecker, and I. D.    Hasday. 1994. Altered cytokine regulation in the lungs of cigarette    smokers. Am J Respir Crit Care Med 150(3):696-703.-   26. Ohta, T., N. Yamashita, M. Maruyama, E. Sugiyama, and M.    Kobayashi. 1998. Cigarette smoking decreases interleukin-8 secretion    by human alveolar macrophages. Respir Med 92(7):922-7.-   27, Suzuki, N., S. Wakisaka, Y. Takeba, S. Mihara, and T.    Sakane. 1999. Effects of cigarette smoking on Fas/Fas ligand    expression of human lymphocytes. Cell Immunol 192(1):48-53.-   28. Zia, S., A. Ndoye, V. T. Nguyen, and S. A. Grando. 1997.    Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and    alpha 7 nicotinic receptors modulating calcium metabolism and    regulating adhesion and motility of respiratory epithelial cells.    Res Commun Mol Pathol Pharmacol 97(3):243-62.-   29. Zhang, S., and T. M. Petro. 1996. The effect of nicotine on    murine CD4 T cell responses. Int J Immunopharmacol 18(8-9):467-78.-   30. Bugeon, L., and M. J. Dallman. 2000, Costimulation of T cells,    Am J Respir Crit Care Med 162(4 Pt 2):S164-8.-   31. Green, J. M. 2000. The B7/CD28/CTLA4 T-cell activation pathway.    Implications for inflammatory lung disease. Am J Respir Cell Mol    Biol 22(3):261-4.-   32, Lenschow, D. J., T. L. Walunas, and J. A. Bluestone. 1996.    CD28/B7 system of T cell costimulation. Annu Rev Immunol 14:233-58.-   33, Walunas, T. L. and J. A. Bluestone. 1998. CTLA-4 regulates    tolerance induction and T cell differentiation in vivo. J Immunol    160(8):3855-60,-   34. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S.    Linsley, G. J. Freeman, J. M. Green, C. B. Thompson, and J. A.    Bluestone. 1994. CTLA-4 can function as a negative regulator of T    cell activation. Immunity 1(5):405-13.-   35. Israel-Assayag, E., A. Dakhama, S. Lavigne, M. Laviolette,    and Y. Cormier. 1999. Expression of costimulatory molecules on    alveolar macrophages in hypersensitivity pneumonitis. Am J Respir    Crit Care Med 159(6):1830-4.-   36. Israel-Assayag, E., M. Fournier, and Y. Cormier. 1999. Blockade    of T cell costimulation by CTLA4-Ig inhibits lung inflammation in    murine hypersensitivity pneumonitis. J Immunol 163 (12):6794-9.-   37. Larche, M., S. J. Till, B. M. Haselden, J. North, S.    Barkans, C. J. Corrigan, A. B. Kay, and D. S. Robinson, 1998.    Costimulation through CD86 is involved in airway antigen-presenting    cell and T cell responses to allergen in atopic asthmatics. J    Immunol 161(11):6375-82.-   38, Mathur, M., K. Herrmann, Y, Qin, F. Gulmen, X. Li, R.    Krimins, J. Weinstock, D. Elliott, J. A. Bluestone, and P.    Padrid. 1999. CD28 interactions with either CD80 or CD86 are    sufficient to induce allergic airway inflammation in mice. Am J    Respir Cell Mol Biol 21(4):498-509.-   39. Nicod, L. P., and P. Islet 1997. Alveolar macrophages in    sarcoidosis coexpress high levels of CD86 (B7.2), CD40, and CD30L.    Am J Respir Cell Mol Biol 17(1):91-6.-   40. Kesingland, A. C., C. T. Gentry, M. S. Panesar, M. A.    Bowes, J. M. Vernier, R. Cube, K. Walker, and L. Urban. 2000.    Analgesic profile of the nicotinic acetylcholine receptor agonists,    (+)-epibatidine and ABT-594 in models of persistent inflammatory and    neuropathic pain. Pain 86(1-2):113-8.-   41, Mellon, R. D., and B. M. Bayer. 1999. The effects of morphine,    nicotine and epibatidine on lymphocyte activity and    hypothalamic-pituitary-adrenal axis responses. J Pharmacol Exp Ther    288(2):635-42.-   42. Yokotani, K., M. Wang, S. Okada, Y. Murakami, and M    Hirata. 2000. Characterization of nicotinic acetylcholine    receptor-mediated noradrenaline release from the isolated rat    stomach. Eur J Pharmacol 1402(3):223-9.-   43. Yost, C. S., and B. D. Winegar. 1997, Potency of agonists and    competitive antagonists on adult- and fetal-type nicotinic    acetylcholine receptors. Cell Mol Neurobiol 17(1):35-50.-   44. Fecho, K., K. A. Maslonek, L. A. Dykstra, and D. T. Lysle. 1993.    Alterations of immune status induced by the sympathetic nervous    system: immunomodulatory effects of DMPP alone and in combination    with morphine. Brain Behav Immun 7(3):253-70.-   45. Thompson, D. C., R. J. Altiere, and L. Diamond. 1990. Nicotinic    agonist modulation of feline bronchomotor tone, Clin Exp Pharmacol    Physiol 17(2):83-97.-   46. Barnes P J, 2001. Future Advances in COPD Therapy. Respiration    68(5):441-8.-   47. Lasky J A and Ortiz, L A. 2001. Antifibrotic therapy for the    treatment of pulmonary fibrosis. Am J Med Sci 322(4):213-21.-   48. Baron, S. A. 1996. Beneficial effects of nicotine and cigarette    smoking: the real, the possible and the spurious. Br Med Bull    52(1):58-73.-   49, Waldium, H. L., O. G. Nilsen, T. Nilsen, H. Rorvik, V.    Syversen, A. K. Sanvik, O. A. Haugen, S. H. Torp, and E.    Brenna. 1996. Long-term effects of inhaled nicotine. Life Sci    58(16):1339-46.-   50, Warren, C. P. 1977. Extrinsic allergic alveolitis: a disease    commoner in non-smokers. Thorax 32(5):567-9.-   51, Cormier, Y., G. M. Tremblay, M. Fournier, and E.    Israel-Assayag. 1994. Long-term viral enhancement of lung response    to Saccharopolyspora rectivirgula. Am J Respir Crit Care Med 149(2    Pt 1):490-4.-   52. Gudmundsson, G., and G. W. Hunninghake. 1997. Interferon-gamma    is necessary for the expression of hypersensitivity pneumonitis. J    Clin Invest 99(10):2386-90.-   53. Denis, M., M. Bedard, M. Laviolette, and Y. Cormier, 1993. A    study of monokine release and natural killer activity in the    bronchoalveolar lavage of subjects with farmer's lung. Am Rev Respir    Dis 147(4):934-9.-   54, Wahlstrom, J., K. Katchar, H. Wigzell, O. Olerup, A. Eklund,    and J. Grunewald. 2001. Analysis of intracellular cytokines in    cd4(+) and cd8(+) lung and blood t cells in sarcoidosis. Am J Respir    Crit Care Med 163(1):115-21.-   55. Cohn, L., C. Herrick, N. Niu, R. Horner, and K. Bottomly. 2001.    IL-4 promotes airway eosinophilia by suppressing EN-gamma    production: defining a novel role for IFN-gamma in the regulation of    allergic airway inflammation. J Immunol 166(4):2760-7.-   56. Laliberte R., Rouabhia M, Bosse M, Chakir J. 2001. Decreased    capacity of asthmatic bronchial fibroblasts to degrade collagen.    Matrix Biol January; 19(8):743-53.-   57. Boulet, L. P., H. Turcotte, M. Laviolette, F. Naud, M. C.    Bernier, S. Martel, and J. Chakir. 2000, Airway hyperresponsiveness,    inflammation, and subepithelial collagen deposition in recently    diagnosed versus long-standing mild asthma, Influence of inhaled    corticosteroids. Am J Respir Crit Care Med 162(4 Pt 1):1308-13.-   58. Dempsey, O. J. 2000. Leukotriene receptor antagonist therapy.    Postgrad Med J 76(902):767-73.-   59. Buss; W. W. 1998. Leukotrienes and inflammation. Am J Respir    Crit Care Med 157(6 Pt 2):S210-3; discussion 5247-8.-   60. Zisman, D. A., J. P. Lynch, G. B. Toews, E. A. Kazerooni, A.    Flint, and F. J. Martinez. 2000. Cyclophosphamide in the treatment    of idiopathic pulmonary fibrosis: a prospective study in patients    who failed to respond to corticosteroids. Chest 117(6):1619-26.-   61. Redington, A. E. 2000. Fibrosis and airway remodelling. Clin Exp    Allergy 30 Suppl 1:42-5.-   62. Frew, A. J., and Plummeridge M J. 2001. Alternative agents in    asthma. J Allergy Clin Immunol 108(1):3-10.

The invention claimed is:
 1. A compound of formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N, Ya is one or more substituent selected from hydrogen,halogen, amidino, amido, azido, cyano, guanido, hydroxyl, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, and aralkyl, n is 2, J is a counter ion.2. The compound as defined in claim 1, wherein R₁ and R₂ areindependently selected from methyl, ethyl, n-propyl, or i-propyl; Xa isCH; Ya is hydrogen; n is 2; J is a halogen.
 3. The compound as definedin claim 1, having the formula:

wherein R₁ and R₂ are independently selected from methyl, ethyl,n-propyl, or i-propyl; Ya is hydrogen; J is a halogen.
 4. The compoundas defined in claim 1, having the formula:


5. The compound as defined in claim 1, wherein R₁ and R₂ areindependently alkyl of 1 to 10 carbon atoms, Xa is CH or N, Ya is one ormore substituent selected from hydrogen, halogen, cyano, hydroxyl, alkylof 1 to 6 carbon atoms and alkoxy of 1 to 6 carbon atoms; n is
 2. 6. Thecompound as defined in claim 1, wherein R₁ and R₂ are independentlyalkyl of 1 to 10 carbon atoms, Xa is CH or N, Ya is hydrogen or halogen,n is
 2. 7. The compound as defined in claim 1, wherein R₁ and R₂ areindependently selected from methyl, ethyl, n-propyl, or i-propyl, Xa isCH or N, Ya is hydrogen or halogen, n is
 2. 8. The compound as definedin claim 7, wherein Ya is hydrogen, and Xa is CH.
 9. The compound asdefined in claim 8, wherein J is fluoride, chloride, bromide, iodide,sulfate or sulfonate.
 10. The compound as defined in claim 1, having theformula:

wherein J is a sulfonate.
 11. A pharmaceutical composition comprising acompound of formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N, Ya is one or more substituent selected from halogen,amidino, amido, azido, cyano, guanido, hydroxyl, nitroso, urea, sulfate,sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl, acyloxy,alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthioof 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms, alkanol of 1to 6 carbon atoms, and aralkyl, n is 2, J is a counter ion; and apharmaceutically acceptable excipient.
 12. A pharmaceutical compositionas defined in claim 11, further comprising one or more therapeutic agentselected from a bronchodilator, anti-inflammatory therapy, a leukotrienereceptor antagonist and phosphodiesterase inhibitors.
 13. Thepharmaceutical composition of claim 11, wherein in said compound, R₁ andR₂ are independently alkyl of 1 to 10 carbon atoms, Xa is CH or N, Ya isone or more substituent selected from hydrogen, halogen, cyano,hydroxyl, alkyl of 1 to 6 carbon atoms and alkoxy of 1 to 6 carbonatoms, n is
 2. 14. The pharmaceutical composition of claim 11, whereinin said compound, R₁ and R₂ are independently alkyl of 1 to 10 carbonatoms, Xa is CH or N, Ya is hydrogen or halogen, n is
 2. 15. Thepharmaceutical composition of claim 11, wherein in said compound, R₁ andR₂ are independently selected from methyl, ethyl, n-propyl, or i-propyl,Xa is CH or N, Ya is hydrogen or halogen, n is
 2. 16. The pharmaceuticalcomposition of claim 15, wherein in said compound, Ya is hydrogen, andXa is CH.
 17. The pharmaceutical composition as defined in claim 16wherein J is fluoride, chloride, bromide, iodide, sulfate or sulfonate.18. The pharmaceutical composition of claim 11, wherein said compoundhas the formula:

wherein J is a sulfonate.
 19. A compound of formula:

wherein R₁ and R₂ are independently lower alkyl of 1 to 10 carbon atoms,Xa is CH or N, Ya is one or more substituent selected from hydrogen,halogen, amidino, amido, azido, cyano, guanido, hydroxyl, nitroso, urea,sulfate, sulfite, sulfonate, sulphonamide, phosphate, phosphonate, acyl,acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms,alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6 carbon atoms,alkanol of 1 to 6 carbon atoms, and aralkyl, n is 2, J is a sulfonate.20. The compound as defined in claim 19, wherein R₁ and R₂ areindependently alkyl of 1 to 10 carbon atoms, Xa is CH or N, Ya is one ormore substituent selected from hydrogen, halogen, cyano, hydroxyl, alkylof 1 to 6 carbon atoms and alkoxy of 1 to 6 carbon atoms; n is
 2. 21.The compound as defined in claim 19, wherein R₁ and R₂ are independentlyalkyl of 1 to 10 carbon atoms, Xa is CH or N, Ya is hydrogen or halogen,n is
 2. 22. The compound as defined in claim 19, wherein R₁ and R₂ areindependently selected from methyl, ethyl, n-propyl, or i-propyl, Xa isCH or N, Ya is hydrogen or halogen, n is 2.